EP2725104B1 - Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité - Google Patents

Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité Download PDF

Info

Publication number
EP2725104B1
EP2725104B1 EP12803011.1A EP12803011A EP2725104B1 EP 2725104 B1 EP2725104 B1 EP 2725104B1 EP 12803011 A EP12803011 A EP 12803011A EP 2725104 B1 EP2725104 B1 EP 2725104B1
Authority
EP
European Patent Office
Prior art keywords
protein
scfv
hyhel
arginine
buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP12803011.1A
Other languages
German (de)
English (en)
Other versions
EP2725104A4 (fr
EP2725104A1 (fr
Inventor
Daisuke Ejima
Haruna Sato
Kouhei Tsumoto
Masayo Date
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ajinomoto Co Inc
Original Assignee
Ajinomoto Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co Inc filed Critical Ajinomoto Co Inc
Publication of EP2725104A1 publication Critical patent/EP2725104A1/fr
Publication of EP2725104A4 publication Critical patent/EP2725104A4/fr
Application granted granted Critical
Publication of EP2725104B1 publication Critical patent/EP2725104B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for producing a multimeric protein consisting of a monomeric protein obtained by fusing a protein having an immunoglobulin fold structure to a protein that can serve as a subunit structure.
  • Therapeutic antibodies have been successful because of their high therapeutic effect and low risk of side effects, but now have a problem of high medical fees due to the high production cost. There are other problems that the therapeutic effect of such antibodies is limited to a relatively narrow range of diseases such as leukemia and autoimmune diseases, and the antibodies are not so effective against solid cancers and the like in which the distribution of a drug to the tissues is limited.
  • Non Patent Literatures 1 to 3 After the production of antibody fragments in Escherichia coli became possible in 1988 (Non Patent Literatures 1 to 3), the development of modified antibodies (antibody fragments) having a high tissue distribution property and a lower molecular weight has been continued. Many antibody fusion proteins have been proposed, which are obtained by fusing an antibody fragment to an anticancer agent, toxin, radioisotope, prodrug activating enzyme, or the like to obtain a higher cancericidal effect.
  • Non Patent Literature 4 multivalent low-molecular-weight modified antibodies and antibody fusion proteins
  • Non Patent Literature 7 A protein fused to streptavidin enables drug pre-targeting using a biotin molecule, which binds to streptavidin with a high affinity.
  • the fusion protein is one of the most promising multivalent fusion proteins (Non Patent Literature 7).
  • a problem thereof is that when a multivalent fusion protein is produced using a microorganism such as Escherichia coli, insoluble proteins are formed in cells of the microorganism in many cases.
  • the insoluble proteins in cells of the microorganism have lost the activity and/or stability. Accordingly, if such proteins are used as a pharmaceutical preparation or the like, refolding operations for reconstructing the active structure are additionally required.
  • Kipriyanov et al. studied the affinity enhancement of a recombinant antibody, made by forming complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion protein (Non Patent Literature 11).
  • the complexes are made by expressing the fusion protein in Escherichia coli, solubilising the protein with a solution containing guanidine hydrochloride, refolding by dialysis against TEA buffer, and isolating the resulting tetrameric complex.
  • the present inventors have previously proposed a method (refolding method) for restoring a higher-order structure of a native state of a protein (denatured protein) which has lost its activity and/or stability as having become insoluble or lost a higher-order structure thereof (Patent Literature 1).
  • the protein structure can be effectively reconstructed by solubilizing a denatured and precipitated protein at pH 6.5 to 9.0 using a 1 to 3% predetermined aqueous solution of an acyl glutamic acid surfactant, and then lowering the concentration of the surfactant in the solubilized solution down to 0.02 to 0.5% using an arginine buffer.
  • Patent Literature 1 International Patent Application Publication No. WO2009/136568
  • An object of the present invention is to provide a novel method for producing a multivalent fusion protein, particularly a multimeric protein containing an immunoglobulin fold structure in a subunit structure.
  • a fusion protein having an immunoglobulin fold structure which has been solubilized with lauroyl-L-Glu, stably continues to be dissolved without forming a multimeric structure even if the concentration of lauroyl-L-Glu is lowered down to 0.02 to 0.5%. Further, the inventors have discovered that while the fusion protein is being dissolved in the solution of lauroyl-L-Glu, only a subunit structure is formed, and subsequently the solution is replaced with a specific buffer, so that a multimeric structure of the fusion protein can be formed.
  • the present invention provides a method for producing a multimeric protein comprising the steps of:
  • the present invention makes it possible to produce a multivalent fusion protein of a multimeric structure having multiple affinity sites for a target antigen, by fusing a protein having an immunoglobulin fold structure to a different protein that can serve as a subunit structure, and utilizing the properties of the fusion protein to form the multimeric structure.
  • a monomeric protein composing a multimeric protein in the present invention is obtained by fusing a protein having an immunoglobulin fold structure to a different protein that can serve as a subunit structure.
  • the self-assembling and self-binding properties of this different protein make it possible to form a multimeric structure, and produce a multivalent fusion protein of a multimeric structure having multiple affinity sites for a target antigen.
  • the protein having an immunoglobulin fold structure include an anti-hen lysozyme antibody (for example, HyHEL-10, D1.3), an anti-TNFa antibody (infliximab, adalimumab, golimumab), an anti-HER2 antibody (trastuzumab), an anti-CD20 antibody (rituximab), an anti-VEGF antibody (bevacizumab), an anti-EGFR antibody (cetuximab, panitumumab), and the like.
  • an anti-hen lysozyme antibody for example, HyHEL-10, D1.3
  • an anti-TNFa antibody infliximab, adalimumab, golimumab
  • an anti-HER2 antibody to stauzumab
  • an anti-CD20 antibody rituximab
  • an anti-VEGF antibody bevacizumab
  • an anti-EGFR antibody cetuximab, panitumumab
  • the different protein that can serve as a subunit structure includes streptavidin, a human p53-derived tetrameric ⁇ -helix structure, a leucine zipper structure, functional fragments thereof, and the like.
  • a functional fragment refers to a protein fragment that can serve as a subunit structure, and an example thereof includes core streptavidin.
  • the monomeric protein used in the present invention specifically includes a fusion protein between scFv and streptavidin, and the like. More specifically, the monomeric protein includes a fusion protein between HyHEL-10 scFv and streptavidin, a fusion protein between D1.3 scFv and streptavidin, a fusion protein between HyHEL-10 scFv and core streptavidin, and the like.
  • the monomeric protein used in the present invention has an insoluble granular form in cells of a microorganism.
  • a "monomeric multimeric protein” is simply stated, it is sometimes intended to mean a protein having an insoluble granular form in cells of a microorganism, the protein obtained by fusing a protein having an immunoglobulin fold structure to a different protein that can serve as a subunit structure.
  • the monomeric protein used in the present invention can be produced by constructing a production system for the target monomeric protein according to methods known in the art, for example, methods described in Ueda et al, Gene. 1993, 129, 129-34 ., Lin, Y. et al, Cancer Res. 2006, 66, 3884-3892 ., Fischmann, T. O. et al, J. Biol. Chem. 1991, 266, 12915-12920 ., and Lin, Y. et al, Cancer Res. 2006, 66, 3884-3892 ., and subsequently culturing and collecting bacterial cells according to a conventional method.
  • a surfactant used to solubilize the monomeric protein is lauroyl glutamic acid (lauroyl-Glu) or a salt thereof.
  • the lauroyl-Glu may have any one of a D form, an L form, and a DL form. Lauroyl-L-Glu is preferable.
  • the lauroyl-Glu or the salt thereof is used in the form of preferably a 1 to 10% aqueous solution thereof, more preferably a 1 to 8% aqueous solution thereof. Within these concentration ranges, it is possible to sufficiently increase the efficiency of solubilizing the monomeric protein while keeping a dilution rate in the next operation at an appropriate level.
  • the pH of the aqueous solution at 25°C can be selected to be a moderate condition of preferably pH 6.5 to 9.0, more preferably pH 7.0 to 8.8, in accordance with properties of the monomeric protein.
  • pH can be measured by a pH meter equipped with a pH electrode.
  • the pH adjustment can be carried out using an alkali, such as sodium hydroxide.
  • a buffer may be used as the aqueous solution.
  • a solution is obtained, in which the monomeric protein is solubilized.
  • the monomeric protein may be added to the lauroyl-Glu aqueous solution, or the lauroyl-Glu aqueous solution may be added to the monomeric protein.
  • the mixture may be left alone normally at 5 to 40°C, preferably at 15 to 40°C. These ranges are preferable because cleavage of the monomeric protein due to a chemical reaction and modifications such as oxidation can be suppressed to the minimum.
  • the period of time for the leaving is normally 10 minutes to 3 hours, preferably 10 minutes to 1 hour. These ranges are preferable because cleavage of the monomeric protein due to a chemical reaction and modifications such as oxidation can be suppressed to the minimum.
  • the mixture may be stirred during the contact.
  • the protein is solubilized or not, for example, by visual examination of turbidity, a UV absorption spectrum method at around 280 nm, or the like.
  • step (B) the solution obtained in step (B), in which the monomeric protein is solubilized, is diluted with a buffer containing arginine or an arginine derivative as an additive at a dilution rate of approximately several tens to 150, and maintained in situ until the monomeric protein restores a higher-order structure of the native state.
  • concentration of the lauroyl-Glu after the dilution is preferably 0.01 to 0.5%, further preferably 0.02 to 0.3%.
  • the dilution may be carried out in an appropriately-selected form of single stage, multi stage (step gradient), or linear gradient.
  • Arginine used as the additive may have an L form or a D form.
  • Arginine may form a salt with an inorganic acid, such as a hydrochloride salt, or a salt with an organic acid, such as acetate salt.
  • the arginine derivative includes arginines having an acyl group with 1 to 6 carbon atoms, such as acetylarginine, N-butyroyl arginine; agmatine with a carboxyl group removed; argininic acid with a hydroxyl group introduced in place of ⁇ -amino group .
  • the arginine derivative is preferably acylated arginine, more preferably N-butyroyl arginine.
  • the additive is most preferably arginine hydrochloride.
  • the buffer sodium phosphate, sodium citrate, tris hydrochloride, or the like may be used.
  • the pH should be suitable for the properties of the protein to be subjected to restoration of the native state, and is generally a neutral pH within pH 6.5 to 9.0. Accordingly, the pH in step (C) should be within this range, and may be different from the pH in step (B).
  • the pH may be adjusted by using, for example, hydrochloric acid, sodium hydroxide, and/or the like.
  • the concentration of the additive is selected each time in accordance with the properties of the protein to be subjected to restoration of the native state.
  • the concentration of the additive is adjusted to make the concentration after the dilution preferably 0.1 to 1.5M, further preferably 0.2 to 1.2 M. These ranges are preferable because restoration of a higher-order structure of the native state can be facilitated.
  • the dilution may be carried out at room temperature, or carried out at 5 to 10°C if the heat stability of a target protein having restored its native state is not sufficiently high.
  • the temperature adjustment may be carried out in an appropriately-selected form of single stage, multi stage (step gradient), or linear gradient.
  • the resultant may be maintained for several hours to several days.
  • the maintenance is desirably carried out for preferably 1 hour to 5 days, more preferably 1.5 hours to 3 days, further preferably 2 hours to 24 hours.
  • the dilution may be carried out gradually.
  • the concentration of the protein after dilution in the last stage is kept to be 0.01 to 1.0 mg/ml, preferably 0.01 to 0.5 mg/ml, by condensation using an ultrafiltration membrane, for example, after the dilution in the last stage in order to cancel out the dilution rate.
  • the target protein contains a Fab
  • such a protein concentration facilitates formation of disulfide bonds among heavy and light chains composing the Fab.
  • a solvent of a solution obtained in step (C) is replaced with a buffer.
  • the lauroyl-Glu is removed from the system.
  • the replacement with the buffer is carried out using a method selected from the group consisting of
  • the buffer used in the present invention is a pH buffer.
  • a strong acid for example, hydrochloric acid, sulfuric acid, formic acid, or the like
  • a weakly basic solution such as tris(hydroxymethyl)aminomethane
  • a dilute solution of a strong alkali for example, sodium hydroxide, lithium hydroxide, or the like
  • the buffer may contain arginine or an arginine derivative, or may contain an inorganic salt, such as NaCl, or a chelating agent, such as EDTA.
  • concentration is for example 0.001 to 1.2 M.
  • the pH of the buffer is preferably 6 to 9.
  • the monomeric protein forms a multimeric protein by UV absorption, static light-scattering method, or the like.
  • a phosphate buffer solution Prior to the dilution with the arginine or arginine derivative buffer, some proteins may be mixed with a phosphate buffer solution or the like and left alone. Specifically, between step (B) and step (C) described above, a phosphate buffer is added to make the concentration of the surfactant 2 to 5%, and thereafter left alone at preferably 5 to 40°C for preferably 10 minutes to 1 hour. Thereby, the percentage of the monomeric protein extracted is increased, and the solubility can be further increased. As a result, the percentage of restoration of a higher-order structure of the protein can be further increased.
  • the pH of the solution thus obtained at 25°C should be in a range from pH 6.5 to 9.0.
  • disulfide bond there may be a disulfide bond within a single molecule. It is preferable to facilitate formation of such disulfide bonds by a redox reaction of the proteins because the percentage of refolding is further improved.
  • the redox reaction may be carried out by adding a redox reagent which facilitates a thiol-disulfide exchange reaction and thereby allows formation of an intramolecular or intermolecular disulfide bond (for example, a mixture of oxidized glutathione (GSSG) and reduced glutathione (GSH), a mixture of cystine and cysteine, a mixture of cystamine and cysteamine, a mixture of oxidized glutathione or cystine and mercaptoethanol, or the like)), or a copper ion which facilitates air oxidation, or may be carried out by changing the redox potential of the protein. It is preferable to use a redox reagent.
  • a redox reagent which facilitates a thiol-disulfide exchange reaction and thereby allows formation of an intramolecular or intermolecular disulfide bond
  • GSSG oxidized glutathione
  • GSH reduced glutathione
  • the redox reaction may be carried out anytime after step (B) described above.
  • the redox reaction may be carried out in step (C) described above by adding a redox reagent together with an additive to the solution obtained in step (A), or may be carried out after the diluted solution is obtained in step (C) described above by adding a redox reagent to the diluted solution.
  • the concentration of the redox reagent or copper ion is adjusted to an appropriate concentration for each protein to be subjected to restoration of the native state.
  • the pH at 25°C in this stage should be in a range from pH 6.5 to 9.0.
  • the pH may be adjusted by using, for example, hydrochloric acid, sodium hydroxide, and/or the like.
  • the temperature of the solution may be in the same range as the temperature of the solution obtained in step (B) or in the same range as the temperature of the solution obtained in step (C).
  • approximately 5 to 48°C is preferable.
  • the resultant may be left alone at 5 to 48°C for approximately 1 hour to 5 days (120 hours).
  • the percentage of refolding is at least 10%, and reaches 30% in many cases.
  • the protein which has restored the higher-order structure can be purified by a normal method, for example, ultrafiltration, dialysis, ion-exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, affinity chromatography, and the like.
  • the present inventors have reported in the previous application (International Patent Application Publication No. WO2009-136568 ) that a protein structure can be effectively reconstructed by solubilizing a denatured protein at pH 6.5 to 9.0 using a 1 to 3% predetermined acyl glutamic acid surfactant, and then diluting using an arginine buffer to lower the concentration of the surfactant down to 0.02 to 0.5%.
  • a multimeric protein has several subunits associated with each other by a non-covalent bond. Nevertheless, even if a multimeric protein is solubilized at a predetermined pH using a predetermined acyl glutamic acid surfactant (lauroyl-L-Glu) and then diluted with an arginine buffer, refolding cannot be completed only by these operations. Although not bound by any theory, it is assumed that lauroyl-L-Glu inhibits the association of the subunits.
  • the present invention has been made by taking advantage of this phenomenon.
  • the present invention is based on the following assumption. Specifically, at first, only formation of a subunit structure is allowed to progress by inhibiting the association of subunits using lauroyl-L-Glu, and thereafter lauroyl-L-Glu considered to be a factor of inhibiting the association is replaced with a buffer and removed. As a result, the subunits are associated with each other, so that the refolding is completed.
  • multimeric protein thus produced, therapeutic drugs, reagent for clinical tests, reagents for researches, and so forth can be obtained against various diseases such as cancers, immune diseases, and lifestyle diseases.
  • These pharmaceutical compositions may include an excipient, a carrier, or the like in addition to the multimeric protein obtained by the method of the present invention.
  • a production system was constructed for a fusion protein (HyHEL-10 scFv SA full length) between a single-chain antibody fragment of variable region (HyHEL-10 scFv) and full length streptavidin (SA full length) using an Escherichia coli BL21 strain (DE3) as a production host ( Ueda et al, Gene. 1993, 129, 129-34 ., Lin, Y. et al, Cancer Res. 2006, 66, 3884-3892 .).
  • HyHEL-10 scFv SA full length was accumulated in the form of insoluble granules in cells of Escherichia coli.
  • the cells were collected, suspended in 20 mM tris hydrochloride and 0.5 M NaCl at pH 8.1, and broken by ultrasonic disintegration.
  • the obtained suspension was subjected to centrifugation under conditions of 6000 g and 30 minutes to recover a precipitate containing insoluble granules of HyHEL-10 scFv SA full length.
  • the recovered precipitate was sequentially washed with a 2% Triton X-100 aqueous solution and acetone. Thereafter, Triton X-100 was completely removed by washing with purified water (Milli Q water). The centrifugation operation was performed under the above conditions again, and a precipitate containing the insoluble granules was obtained.
  • a precipitate containing insoluble granules of D1.3 scFv SA full length was obtained in the same manner as in Reference Example 1, except that a production system was constructed for a fusion protein (D1.3 scFv SA full length) between full length streptavidin (SA full length) and D1.3 scFv, which was used as a single-chain antibody fragment of variable region in place of HyHEL-10 scFv in Reference Example 1 ( Fischmann, T. O. et al, J. Biol. Chem. 1991, 266, 12915-12920 ., Lin, Y. et al, Cancer Res. 2006, 66, 3884-3892 .).
  • a 5% lauroyl-L-Glu solution (20 mM sodium phosphate, pH 8.5) was prepared.
  • the precipitate in each of the tubes was solubilized by adding 0.2 ml of the solution into the tubes 1, 0.25 ml into the tube 2, 0.3 ml into the tube 3, and 0.35 ml into the tube 4. After placed in a vortex mixer at room temperature, the tubes 1 to 4 were gently centrifuged (10000 rpm, 1 minute) to remove formed foams.
  • 20 mM sodium phosphate (pH 7) was added and filled to 0.5 ml. The final concentrations of lauroyl-L-Glu in the tubes 1 to 4 were adjusted to 2.0%, 2.5%, 3.0%, and 3.5%, respectively.
  • a 2% lauroyl-L-Glu solution (20 mM sodium phosphate, pH 8.5) was prepared.
  • (2) The solution obtained in (1) was used and added to 100 mg of the precipitate obtained in Reference Example 1 to solubilize the precipitate in the same manner as in Experiment 1.
  • (3) A diluting solution containing arginine hydrochloride was prepared.
  • (4) The solution obtained in (2) was diluted 100 fold using the diluting solution prepared in (3).
  • prepared was 30 ml of 0.02% lauroyl-L-Glu, 0.8 M arginine hydrochloride, 1 mM EDTA, 80 mM tris hydrochloride, 1 mM reduced glutathione, and 1 mM oxidized glutathione, with pH 8.0.
  • the concentration of the protein was adjusted to 0.02 mg/ml.
  • the solution obtained in (4) was maintained, then concentrated 3 fold in concentration using a centrifugal ultrafiltration membrane, and adjusted to 10 ml.
  • (6) After 40 ⁇ l of the 3-fold concentrated solution obtained in (5) was subjected to centrifugation at 14000 rpm (18700 ⁇ g) for 10 minutes, 10 ⁇ l of the resulting supernatant was subjected to gel filtration HPLC in the same manner as in Experiment 1 to quantify HyHEL-10 scFv SA full length having a tetrameric structure formed.
  • HyHEL-10 scFv SA full length multimeric proteins can be recovered by diluting to make the concentration of arginine hydrochloride 0.4 to 1.2 M after the solubilization.
  • the concentration of the protein was adjusted to 0.05 mg/ml.
  • each of the solutions obtained in (3) was maintained, then concentrated 3 fold in concentration using a centrifugal ultrafiltration membrane, and adjusted to 2.5 ml.
  • Each 2.5 ml of the 3-fold concentrated solutions obtained in (4) was loaded onto a PD-10 column equilibrated in advance with a buffer (50 mM tris hydrochloride, 0.4 M arginine hydrochloride, 1 mM EDTA, pH 8.0), and subsequently deployed using 3.0 ml of the same buffer. Thereby, the total amount of the concentrated solution was recovered.
  • HyHEL-10 scFv SA full length having a tetrameric structure formed can be recovered even if the concentrations of reduced glutathione and oxidized glutathione vary from none to 5 mM. This revealed that when a specific combination of reduced and oxidized glutathione concentrations is necessary depending on a protein, an optimal concentration combination can be selected as appropriate.
  • the salt added to the buffer was NaCl or arginine hydrochloride
  • the HyHEL-10 scFv SA full length multimeric proteins were successfully recovered. It was found that when arginine hydrochloride is used as a salt to be added, the amount of a tetrameric protein recovered is increased in comparison with a case where NaCl is used. It was found that when NaCl is used as a salt to be added, the recovery amount is increased if the pH is increased from 6.6 to 7.6.
  • HyHEL-10 scFv SA full length multimeric proteins can be recovered by setting the pH of the buffer at 6.5 to 8.4.
  • HyHEL-10 scFv SA full length multimeric protein is recovered by setting the concentration of arginine hydrochloride added to the buffer at 0.05 to 0.8 M. It was found that the recovery amount is high when the concentration of arginine hydrochloride added is 0.2 to 0.8 M.
  • a 50 mM phosphoric acid solution pH 6.2
  • 200 mM NaCl 50 mM phosphoric acid solution
  • a production system was constructed for a fusion protein (HyHEL-10 scFv SAcore) between a single-chain antibody fragment of variable region (HyHEL-10 scFv) and naturally occurring wildtype core streptavidin (natural core Streptavidin) using an Escherichia coli BL21 strain (DE3) as a production host.
  • the amino acid sequencing of the natural core Streptavidin followed a published report ( Takeshi Sano, et al. The journal of biological chemistry 270, 47, 28204-28209 (1995 ).
  • the production bacterium thus constructed was cultured according to an ordinary method in an LB medium at 37°C with shaking.
  • the absorbance at 660 nm after the culturing for several hours was confirmed to be approximately 0.8.
  • IPTG isopropyl-p-thiogalactopyranoside
  • the culturing was further continued for 4 to 5 hours, so that HyHEL-10 scFv SAcore was accumulated in the form of insoluble granules in the cells of Escherichia coli.
  • the cells were recovered from 250 ml of the culture solution, suspended in 20 ml of 20 mM tris hydrochloride, 30 mM NaCl, 5 mM EDTA at pH 7.5, and broken by ultrasonic disintegration at 60 W in an ice bath for 3 minutes.
  • the obtained disrupted cell solution was subjected to centrifugation at 5°C at 4400 g for 10 minutes, and 20 ml of of the obtained precipitate was again suspended in 20 mM tris hydrochloride, 30 mM NaCl, and 5 mM EDTA at pH 7.5.
  • the suspension was subjected to centrifugation at 5°C at 6500 g for 10 minutes to obtain a precipitate.
  • the same operation was further repeated one more time.
  • 1.11 g of a precipitate containing insoluble granules of HyHEL-10 scFv SAcore was obtained.
  • mixture solutions each 100 ⁇ l, were prepared in which the final concentration of a hen egg white lysozyme (Code No. L-6876, manufactured by Sigma-Aldrich Corporation) was adjusted to 0.09 ⁇ M, whereas the final concentration of the purified HyHEL-10 scFv SAcore multimeric protein was adjusted to seven levels from 0.0056 to 0.045 pM.
  • the mixture solutions were maintained at 28°C for 30 minutes.
  • HyHEL-10 scFv SAcore multimeric protein has four anti-lysozyme antibody domains (HyHEL-10 scFvs) in a molecule thereof, the molar mixing ratio of lysozyme:HyHEL-10 scFv in these mixture solutions corresponds to 1:0.25 to 1:2.
  • a microbial suspension (Micrococcus lisodeikicus, ATCC4698, SIGMA M-3770, manufactured by Sigma-Aldrich Corporation) was added.
  • the microbial suspension had been adjusted with 50 mM phosphate buffer at pH 6.2, so that the suspension had an absorbance of 1.78 at 540 nm.
  • the absorbance was measured at a measurement wavelength of 540 nm at 28°C every 5 minutes for 1 hour.
  • HyHEL-10 scFv SAcore multimeric protein has four anti-lysozyme antibody domains (HyHEL-10 scFvs) in a molecule thereof
  • the molar mixing ratios of lysozyme: HyHEL-10 scFv in these mixture solutions respectively correspond to 0:1, 0.5:1, and 1:1.
  • the molecular weight was calculated to be 163.4 kDa, 188.1 kDa, and 225.0 kDa, respectively indicating that 0 molecules (no lysozyme), 1.7 molecules, and 4. 3 molecules of the hen egg white lysozyme bound to one molecule of the purified HyHEL-10 scFv SAcore multimeric protein.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (5)

  1. Procédé de production d'une protéine multimère comprenant les étapes consistant à :
    (A) préparer une protéine monomère ayant une forme granulaire insoluble dans de cellules d'un microorganisme, la protéine monomère étant une protéine de fusion d'une protéine ayant une structure pliée d'immunoglobuline et d'une protéine différente ou d'un fragment fonctionnel de celle-ci pouvant servir de structure de sous-unité pour un assemblage avec une ou plusieurs autres structures de sous-unité par liaison non covalente pour former une protéine ayant une structure multimère stable ;
    (B) solubiliser la protéine monomère préparée à l'étape (A) avec une solution aqueuse contenant un quelconque parmi l'acide lauroylglutamique et un sel de celui-ci ;
    (C) diluer une solution obtenue à l'étape (B) dans un tampon contenant un quelconque parmi de l'arginine et un dérivé de l'arginine et des sels d'acides minéraux ou organiques de celle-ci, afin d'abaisser une concentration d'un quelconque parmi l'acide lauroylglutamique et un sel de celui-ci, dans lequel le dérivé d'arginine est choisi dans le groupe comprenant de l'arginine ayant un groupe acyle ayant 1 à 6 atomes de carbone, de l'ester d'arginine butylique, de l'agmatine et de l'acide argininique ; et
    (D) remplacer un solvant d'une solution obtenue à l'étape (C) par un tampon, entraînant l'élimination dudit acide lauroylglutamique et/ou d'un sel de celui-ci, en utilisant un procédé choisi dans le groupe consistant en
    (d1) une chromatographie sur colonne choisie dans le groupe comprenant une chromatographie par filtration sur gel, une chromatographie par échange d'ions, une chromatographie par interaction hydrophobe et une combinaison de celles-ci ;
    (d2) une ultrafiltration ;
    (d3) une dialyse ; et
    (d4) une combinaison de deux ou plus de (d1) à (d3) ; en formant ainsi une structure multimère de la protéine de fusion.
  2. Procédé selon la revendication 1, dans lequel la protéine monomère est une protéine de fusion entre le scFv et la streptavidine.
  3. Procédé selon la revendication 2, dans lequel le scFv est choisi dans le groupe constitué de HyHEL-10 scFv et D1.3 scFv.
  4. Procédé selon la revendication 2 ou 3, dans lequel la streptavidine est un quelconque parmi la streptavidine de pleine longueur et un fragment fonctionnel de celle-ci.
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel le dérivé d'arginine est le chlorhydrate d'arginine.
EP12803011.1A 2011-06-23 2012-06-25 Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité Active EP2725104B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011139837 2011-06-23
PCT/JP2012/066121 WO2012176919A1 (fr) 2011-06-23 2012-06-25 Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité

Publications (3)

Publication Number Publication Date
EP2725104A1 EP2725104A1 (fr) 2014-04-30
EP2725104A4 EP2725104A4 (fr) 2014-11-12
EP2725104B1 true EP2725104B1 (fr) 2019-08-21

Family

ID=47422745

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12803011.1A Active EP2725104B1 (fr) 2011-06-23 2012-06-25 Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité

Country Status (7)

Country Link
US (1) US10308969B2 (fr)
EP (1) EP2725104B1 (fr)
JP (1) JP5858251B2 (fr)
CN (2) CN103608463A (fr)
IL (1) IL229682B (fr)
IN (1) IN2014CN00436A (fr)
WO (1) WO2012176919A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012176919A1 (fr) 2011-06-23 2012-12-27 味の素株式会社 Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2197210T3 (es) 1994-11-30 2004-01-01 Ajinomoto Co., Inc. Agente antitrombotico y anticuerpos monoclonales contra el factor von willebrand.
US7144991B2 (en) * 1999-06-07 2006-12-05 Aletheon Pharmaceuticals, Inc. Streptavidin expressed gene fusions and methods of use thereof
ES2356149T3 (es) 2003-03-07 2011-04-05 Ajinomoto Co., Inc. Procedimiento para producir transglutaminasa microbiana.
US8084032B2 (en) 2004-01-21 2011-12-27 Ajinomoto Co., Inc. Purification method which prevents denaturation of an antibody
JP4730302B2 (ja) 2004-04-20 2011-07-20 味の素株式会社 タンパク質の製造法
JP5505853B2 (ja) 2007-02-13 2014-05-28 味の素株式会社 微酸性アルギニンを添加剤とするウイルス不活化法
WO2008099898A1 (fr) 2007-02-15 2008-08-21 Ajinomoto Co., Inc. Transglutaminase ayant une liaison disulfure introduite dans celle-ci
CN102089319B (zh) * 2008-05-08 2013-11-13 味之素株式会社 蛋白质的重折叠方法
EP2404999B1 (fr) 2009-03-06 2015-04-29 Ajinomoto Co., Inc. Transglutaminase thermotolérante provenant d'actinomyces
EP2538790B1 (fr) 2010-02-26 2015-07-29 Ajinomoto Co., Inc. Composition antivirale contenant un composé de faible poids moléculaire et d'arginine
WO2012176919A1 (fr) 2011-06-23 2012-12-27 味の素株式会社 Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JEREMY M BERG, JOHN L TYMOCZKO, AND LUBERT STRYER.: "Biochemistry 5th Edition.", 2002, W.H. FREEMAN AND COMPANY, internet https://www.ncbi.nlm.nih.gov/books/NBK21154/, article SECTION 32.2.: "The Immunoglobulin fold consists of a sandwich framework with hypervariable loops" *

Also Published As

Publication number Publication date
WO2012176919A1 (fr) 2012-12-27
JP5858251B2 (ja) 2016-02-10
IN2014CN00436A (fr) 2015-04-03
JPWO2012176919A1 (ja) 2015-02-23
CN103608463A (zh) 2014-02-26
CN107365388A (zh) 2017-11-21
CN107365388B (zh) 2021-05-14
US20140099672A1 (en) 2014-04-10
US10308969B2 (en) 2019-06-04
EP2725104A4 (fr) 2014-11-12
IL229682B (en) 2018-04-30
IL229682A0 (en) 2014-01-30
EP2725104A1 (fr) 2014-04-30

Similar Documents

Publication Publication Date Title
EP3299383A1 (fr) MODIFICATION SPÉCIFIQUE DE L'ANTICORPS PAR UN PEPTIDE DE LIAISON À IgG
Yeboah et al. Elastin‐like polypeptides: A strategic fusion partner for biologics
ES2848861T3 (es) Enzima mutante EndoS
JP5931255B2 (ja) 免疫グロブリン溶液を精製するための方法
EP4159749A2 (fr) Procédé de production d'une molécule contenant des fc avec une chaîne glucidique remodelée
JP5033177B2 (ja) 陽イオン界面活性剤によるタンパク質の精製
JP6419844B2 (ja) Ｃｙｓ連結された抗体−薬物コンジュゲートの精製方法
JP2016523812A (ja) タンパク質のピログルタミン酸形成を増加させるための方法
KR20130131307A (ko) 단백질 정제
EP2725104B1 (fr) Procédé de préparation de protéine multimérique constituée d'une protéine monomérique unissant une protéine ayant une structure pliée d'immunoglobuline et une protéine pouvant prendre une structure de sous unité
JP6231559B2 (ja) 高グリコシル化された持続型ヒト成長ホルモンタンパク質の製造方法及び精製方法
JP5635682B2 (ja) 疎水性相互作用クロマトグラフィー法
Premsukh et al. Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation
CN104854239A (zh) 蛋白质纯化
KR102350592B1 (ko) 히알루로니다제의 정제방법
CA2969981A1 (fr) Methode de fabrication d'une proteine couplee a une molecule de polyethylene glycol
JP2012072091A (ja) 抗体の精製方法
CN114085291B (zh) 一种降低或消除重组蛋白cex酸性峰的方法
KR20200022052A (ko) 설파타아제 단백질을 정제하는 방법
JP5733693B2 (ja) 組換え型ポリペプチドの製造方法
JPWO2011118731A1 (ja) チオスルホナート化合物、タンパク質及び／又はペプチドの可逆的カチオン化剤並びに可溶化方法
JP2020152644A (ja) Ｖｎａｒのリフォールディング方法およびｖｎａｒの製造方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20131210

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20141015

RIC1 Information provided on ipc code assigned before grant

Ipc: C07K 19/00 20060101ALI20141009BHEP

Ipc: C07K 1/113 20060101ALI20141009BHEP

Ipc: C12P 21/02 20060101AFI20141009BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20171222

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20181023

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTC Intention to grant announced (deleted)
INTG Intention to grant announced

Effective date: 20190318

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602012063194

Country of ref document: DE

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1169816

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190915

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: MICHELI AND CIE SA, CH

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191121

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191121

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191223

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191122

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191221

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1169816

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200224

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602012063194

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG2D Information on lapse in contracting state deleted

Ref country code: IS

26N No opposition filed

Effective date: 20200603

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20200625

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200625

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20200630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200625

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200630

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200625

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190821

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230512

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20230502

Year of fee payment: 12

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20230702

Year of fee payment: 12